Osteoclasts are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Antiseptic products have received recent attention as potential therapeutic and preventive drugs in human disease. The purpose of this study was to investigate the effect of the antiseptic cetylpyridinium chloride (CPC) on osteoclast formation using mouse bone marrow-derived macrophages (BMMs). CPC inhibited receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclast formation in a dose-dependent manner without causing cytotoxicity. The mRNA expression of cathepsin K, calcitonin receptor (CTR), and Prdm1 in osteoclasts was reduced by CPC. In experiments to elucidate its mechanism of action, CPC was found to suppress RANKL-induced expression of c-Fos and nuclear factor of activated T cells (NFATc1), transcription factors that are essential for osteoclast differentiation. CPC also inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK) and NF-κB and expression of cyclooxygenase (COX)-2. These results collectively suggest that CPC inhibits osteoclast differentiation by suppressing the activation of ERK and NF-κB and reducing the expression of COX-2, c-Fos, and NFATc1. CPC may therefore be a useful drug in the prevention of bone loss.